A Guide To Freeze Drying for the Laboratory

An Industry Service Publication 2 Foreword Introduction

This booklet has been developed to serve as a basic Freeze drying has been used in a number of guide to the freeze drying process. The information applications for many years, most commonly in the food presented is generic in nature and is the result of and pharmaceutical industries. There are, however, many research and experience by Labconco personnel and other uses for the process including heat-sensitive users of freeze drying equipment. It is our intention to sample preparation, plant material research, the provide a non-biased review of preparation techniques stabilization of living materials such as microbial and freeze drying methods. The purpose of this booklet is cultures, long term storage of HPLC samples, to help you make an informed choice of equipment for preservation of whole animal specimens for museum your laboratory applications. display, restoration of books and other items damaged by water, and the concentration and recovery of reaction products. Specialized equipment is required to create the Our Method conditions conducive to the freeze drying process. This equipment is currently available and can accommodate freeze drying of materials from laboratory scale projects We begin our discussion of freeze drying for the to industrial production. laboratory by examining the three steps in the process: Freeze drying involves the removal of water or other prefreezing, primary drying and secondary drying. Next, solvent from a frozen product by a process called we examine a typical freeze drying cycle and the methods sublimation. Sublimation occurs when a frozen liquid available to facilitate the freeze drying process using goes directly to the gaseous state without passing equipment designed for use by laboratories. Finally, through the liquid phase. In contrast, drying at ambient suggestions to optimize successful results are discussed, temperatures from the liquid phase usually results in including determination of end point, contamination, changes in the product, and may be suitable only for backfilling of dried samples and product stability. some materials. However, in freeze drying, the material A glossary of terms used throughout this booklet to does not go through the liquid phase, and it allows the explain the freeze drying process follows the text, along preparation of a stable product that is easy to use and with a bibliography. aesthetic in appearance. The advantages of freeze drying are obvious. Properly freeze dried products do not need refrigeration, and can be stored at ambient temperatures. Because the cost of the specialized equipment required for freeze drying can be substantial, the process may appear to be an expensive undertaking. However, savings realized by stabilizing an otherwise unstable product at ambient temperatures, thus eliminating the need for refrigeration, more than compensate for the investment in freeze drying equipment.

3 Principles of Freeze Drying vapor pressure of the product compared to the vapor pressure of the ice collector. Molecules migrate from the higher pressure sample to a lower pressure area. Since The freeze drying process consists of three stages: vapor pressure is related to temperature, it is necessary prefreezing, primary drying, and secondary drying. that the product temperature is warmer than the cold Prefreezing: Since freeze drying is a change in state trap (ice collector) temperature. It is extremely from the solid phase to the gaseous phase, material to be important that the temperature at which a product is freeze dried must first be adequately prefrozen. The freeze dried is balanced between the temperature that method of prefreezing and the final temperature of the maintains the frozen integrity of the product and the frozen product can affect the ability to successfully freeze temperature that maximizes the vapor pressure of the dry the material. product. This balance is key to optimum drying. The Rapid cooling results in small ice crystals, useful in typical phase diagram shown in Figure 1 illustrates this preserving structures to be examined microscopically, point. Most products are frozen well below their eutectic but resulting in a product that is more difficult to or glass transition point (Point A), and then the freeze dry. Slower cooling results in larger ice crystals temperature is raised to just below this critical and less restrictive channels in the matrix during the temperature (Point B) and they are subjected to a drying process. reduced pressure. At this point the freeze drying process Products freeze in two ways, depending on the is started. makeup of the product. The majority of products that are subjected to freeze drying consist primarily of water, the Figure 1 solvent, and the materials dissolved or suspended in the water, the solute. Most samples that are to be freeze dried F are which are a mixture of substances that U eutectics S I O CRITICAL freeze at lower temperatures than the surrounding water. N POINT L Liquid E I When the aqueous suspension is cooled, changes occur N IN E Phase L in the solute concentrations of the product matrix. And N IO T A as cooling proceeds, the water is separated from the A IZ R O solutes as it changes to ice, creating more concentrated P A areas of solute. These pockets of concentrated materials Solid V Vapor PRESSURE Phase have a lower freezing temperature than the water. Phase B TRIPLE E POINT N Although a product may appear to be frozen because of LI N TIO all the ice present, in actuality it is not completely frozen MA UBLI C until all of the solute in the suspension is frozen. The S D mixture of various concentration of solutes with the solvent constitutes the eutectic of the suspension. Only TEMPERATURE when all of the eutectic mixture is frozen is the suspension properly frozen. This is called the eutectic temperature. A typical phase diagram. It is very important in freeze drying to prefreeze the Some products such as aqueous sucrose solutions can product to below the eutectic temperature before undergo structural changes during the drying process beginning the freeze drying process. Small pockets of resulting in a phenomenon known as collapse. Although unfrozen material remaining in the product expand the product is frozen below its eutectic temperature, and compromise the structural stability of the freeze warming during the freeze drying process can affect the dried product. structure of the frozen matrix at the boundary of the The second type of frozen product is a suspension that drying front. This results in a collapse of the structural undergoes glass formation during the freezing process. matrix. To prevent collapse of products containing Instead of forming eutectics, the entire suspension becomes increasingly viscous as the temperature is lowered. Finally the product freezes at the glass transition point forming a vitreous solid. This type of product is extremely difficult to freeze dry. Primary drying: Several factors can affect the ability to freeze dry a frozen suspension. While these factors can be discussed independently, it must be remembered that they interact in a dynamic system, and it is this delicate balance between these factors that results in a properly freeze dried product. After prefreezing the product, conditions must be established in which ice can be removed from the frozen product via sublimation, resulting in a dry, structurally intact product. This requires very careful control of the two parameters, temperature and pressure, involved in the freeze drying system. The rate of sublimation of ice A vacuum pump is essential to evacuate the from a frozen product depends upon the difference in environment around the product to be freeze dried.

4 sucrose, the product temperature must remain below a A third component essential in a freeze drying system critical collapse temperature during primary drying. The is energy. Energy is supplied in the form of heat. Almost collapse temperature for sucrose is -32° C. ten times as much energy is required to sublime a gram No matter what type of freeze drying system is used, of water from the frozen to the gaseous state as is conditions must be created to encourage the free flow of required to freeze a gram of water. Therefore, with all water molecules from the product. Therefore, a vacuum other conditions being adequate, heat must be applied to pump is an essential component of a freeze drying the product to encourage the removal of water in the system, and is used to lower the pressure of the form of vapor from the frozen product. The heat must be environment around the product (to Point C). The other very carefully controlled, as applying more heat than the essential component is a collecting system, which is a evaporative cooling in the system can remove warms the cold trap used to collect the moisture that leaves the product above its eutectic or collapse temperature. frozen product. The collector condenses out all Heat can be applied by several means. One method is condensable gases, i.e; the water molecules, and the to apply heat directly through a thermal conductor shelf vacuum pump removes all non-condensable gases. such as is used in tray drying. Another method is to use ambient heat as in manifold drying. Secondary drying: After primary freeze drying is complete, and all ice has sublimed, bound moisture is still present in the product. The product appears dry, but the residual moisture content may be as high as 7-8%. Continued drying is necessary at the warmer temperature to reduce the residual moisture content to optimum values. This process is called isothermal desorption as the bound water is desorbed from the product. Secondary drying is normally continued at a product

temperature higher than ambient but compatible with the sensitivity of the product. All other conditions, such

as pressure and collector temperature, remain the same. Because the process is desorptive, the vacuum should be as low as possible (no elevated pressure) and the collector temperature as cold as can be attained. Secondary drying is usually carried out for approximately 1/3 to 1/2 the time required for primary drying. The collecting system acts as a cold trap to collect moisture leaving the frozen product. It is important to understand that the vapor pressure How Freeze Drying Works of the product forces the sublimation of the water vapor molecules from the frozen product matrix to the collector. The molecules have a natural affinity to move Refer to the phase diagram (Figure 1) and a typical toward the collector because its vapor pressure is lower sublimation cycle (Figure 2). The product is first cooled than that of the product. Therefore, the collector to below its eutectic temperature (Point A). The collector temperature (Point D) must be significantly lower than is cooled to a temperature approximately 20° C cooler the product temperature. As can be noted in Table 1, than the product temperature, generally around -50 to raising the product temperature has more effect on -105° C. The product should be freeze dried at a the vapor pressure differential than lowering the temperature slightly lower than its eutectic or collapse collector temperature. temperature (Point B) since the colder the product, the longer the time required to complete primary drying, and Table 1 the colder the collector temperature required to adequately freeze dry the product. Vapor Pressure (mBar) Temperature (°C) After the product is adequately frozen and the collector temperature achieved, the system is evacuated 6.104 0 using a vacuum pump (Point C). At this point, primary 2.599 -10 drying of the product begins and continues until the 1.034 -20 entire frozen matrix appears dry. Heat input to the 0.381 -30 product may be achieved by several means such as 0.129 -40 increasing the shelf temperature in the case of tray drying, or using a liquid bath for manifold drying. While 0.036 -50 the collector and vacuum pump create the conditions for 0.011 -60 allowing sublimation to occur, heat input is really the 0.0025 -70 driving force behind the whole process. 0.0005 -80 Heat input to the sample can be enhanced by controlling the pressure in the system at some level Vapor Pressure/Temperature Relationships above the ultimate capability of the vacuum pump. Some

5 freeze dryers incorporate vacuum control systems that product, drying becomes more and more difficult. The automatically regulate the pressure to the preset level. water molecules must now travel through the dried This allows additional gas molecules to reside in the portions of the product which impedes their progress. As system thereby improving the conduction of heat to the the drying front moves farther and farther down the sample. This improves the sublimation rate, reducing matrix, the application of heat to the product becomes process time and associated energy costs. Care must be more important (Figure 3). taken to prevent the pressure within the system from Shell freezing as a method of prefreezing the product exceeding the ice vapor pressure of the product or can increase the surface area to volume ratio by melting of the sample may occur. spreading out the frozen product inside the vessel (Figure 4). Shell freezing is accomplished by rotating the vessel in a low temperature bath causing the product to Figure 2 freeze in a thin layer on the inside surface of the vessel. The thickness of the frozen suspension depends on the When primary drying is complete volume of the product in comparison to the size of the product temperature equals shelf temperature vessel. Shell freezing is primarily used in conjunction with manifold drying. ORP RESNEDNOCB UD TC The vacuum system is very important during freeze ET PM LEHS RE drying because the pressure must be maintained at a low METF UTA ERUTAREPMET ER level to ensure adequate water vapor flow from the AREP As drying proceeds product temperature remains below TEMPERATURE ERUT shelf temperature product to the collector. A pressure gauge (commonly A called a vacuum gauge) is used to monitor the pressure D in the system during the drying process. Pressure can be C expressed in several different units which are compared in Table 2. Some gauges measure condensable gases, while others do not. Those gauges that do not measure the condensable gases give an indication of the total PRESSURE pressure in the system. Gauges that do sense the TIME condensable gases indicate a change in pressure during Typical Sublimation Cycle found in system utilizing Tray drying. These sensors can be used as an indication of Dryer with shelves. the rate of drying, as well as the endpoint of the drying process. Heat input to the product must be very carefully controlled especially during the early stages of drying. Figure 4 The configuration of the product container and the volume of the contained product can affect the amount of heat that can be applied. For small volumes of material, evaporative cooling compensates for high levels of heat and drying is accelerated. The volume and configuration of the suspension to be freeze dried often determines how the material is freeze dried. For example, the greater the ratio of the surface area to the volume of the suspension, the faster drying occurs. This is because a greater area for the water molecules to leave the product exists compared to the distance they have to travel to reach the surface of the Build-up frozen matrix. Drying occurs from the top of the product of Frozen and initially the removal of water molecules is efficient. Material However, as the drying front moves down through the Figure 3

Shell freezing can increase the surface area to volume ratio by spreading out the frozen product inside the vessel.

Table 2 Dry Cake Water Microns mm Hg Torr mBar Frozen Sample Vapor Interface 1000 111.33 100 0.1 0.1 0.133 Ambient room temperature provides heat to encourage 10 0.01 0.01 0.03 removal of water vapor from frozen samples when manifold drying. Pressure Relationships

6 Freeze Drying Methods Heat input can be affected by simply exposing the vessels to ambient temperature or via a circulating bath. For some products, where precise temperature control is Three methods of freeze drying are commonly used: required, manifold drying may not be suitable. (1) manifold drying, (2) batch drying, and (3) bulk Several vessels can be accommodated on a manifold drying. Each method has a specific purpose, and the system allowing drying of different products at the same method used depends on the product and the final time, in different sized vessels, with a variety of closure configuration desired. systems. Since the products and their volumes may differ, Manifold Method. In the manifold method, flasks, each vessel can be removed from the manifold separately ampules or vials are individually attached to the ports of as its drying is completed. The close proximity to the a manifold or drying chamber. The product is either collector also creates an environment that maximizes frozen in a freezer, by direct submersion in a low drying efficiency. temperature bath, or by shell freezing, depending on the Batch Method. In batch drying, large numbers of nature of the product and the volume to be freeze dried. similar sized vessels containing like products are placed The prefrozen product is quickly attached to the drying together in a tray dryer. The product is usually prefrozen chamber or manifold to prevent warming. The vacuum on the shelf of the tray dryer. Precise control of the must be created in the product container quickly, and the product temperature and the amount of heat applied to operator relies on evaporative cooling to maintain the the product during drying can be maintained. Generally low temperature of the product. This procedure can only all vials in the batch are treated alike during the drying be used for relatively small volumes and products with process, although some variation in the system can high eutectic and collapse temperatures. occur. Slight differences in heat input from the shelf can Manifold drying has several advantages over batch tray be experienced in different areas. Vials located in the drying. Since the vessels are attached to the manifold front portion of the shelf may be radiantly heated individually, each vial or flask has a direct path to the through the clear door. These slight variations can result collector. This removes some of the competition for in small differences in residual moisture. molecular space created in a batch system, and is most Batch drying allows closure of all vials in a lot at the ideally realized in a cylindrical drying chamber where the same time, under the same atmospheric conditions. The distance from the collector to each product vessel is the vials can be stoppered in a vacuum, or after backfilling same. In a “tee” manifold, the water molecules leaving with inert gas. Stoppering of all vials at the same time the product in vessels farthest from the collector ensures a uniform environment in each vial and uniform experience some traffic congestion as they travel past the product stability during storage. Batch drying is used to ports of other vessels. prepare large numbers of ampules or vials of one product.

Batch drying in a tray dryer permits precise control of product temperature and heat input. Bulk Method. Bulk drying is generally carried out in a tray dryer like batch drying. However, the product is poured into a bulk pan and dried as a single unit. Although the product is spread throughout the entire surface area of the shelf and may be the same thickness as product dried in vials, the lack of empty spaces within the product mass changes the rate of heat input. The heat input is limited primarily to that provided by contact with the shelf as shown in Figure 5. Bulk drying does not lend itself to sealing of product under controlled conditions as does manifold or batch drying. Usually the product is removed from the freeze dry system prior to closure, and then packaged in air In the manifold drying method, flasks are individually tight containers. Bulk drying is generally reserved for attached to the ports of a drying chamber. stable products that are not highly sensitive to oxygen or moisture.

7 Figure 5 numbers of vials are dried in a single chamber. Evidence for contamination may be found by sampling the surfaces of the vials, shelves and collector. The greatest degree of Some heat input at contamination is usually found on the vials and on the edges where drying occurs faster than collector. Although some vial contamination may be due in middle to sloppiness in dispensing the material originally,

contamination on the collector is due to microorganisms traveling from the product to the collector through the vapor stream. The potential for contamination must be considered every time microorganisms are freeze dried, and precautions must be taken in handling material after the freeze dry process is completed. Recognizing that the vials are potentially contaminated, the operator should remove the vials to a safe area such as a laminar flow hood for decontamination. Decontamination of the freeze dry system depends upon the type of freeze dry system used. Some tray dryer systems are designed for In bulk drying, heat is provided primarily through decontamination under pressure using ethylene oxide conduction from shelf. sterilization. Ethylene oxide is considered hazardous, corrosive and detrimental to rubber components. Its use should be carefully monitored. Coupled with the risk of contamination in a freeze dry Determining Drying Endpoints system is the risk of cross contamination when freeze drying more than one product at time. It is not good practice to mix microbiological products in a freeze dry Several means can be used to determine the endpoint system unless some type of bacteriological filter is of primary drying. The drying boundary in batch drying used to prevent the microbial product from leaving the containers has moved to the bottom of the product vial itself. container and inspection reveals that no ice is visible in While freeze drying of corrosive materials does not the product. No visible ice indicates only that drying at necessarily present a risk to the operator, it does present the edges of the container is complete and gives no a risk of damaging the freeze dry system itself. Although indication of the conditions in the center of the product. freeze dry systems are designed using materials that An electronic vacuum gauge can be used to measure resist corrosion and prevent the build up of corrosive condensable gases in the system. When the pressure materials, care should be taken to clean the system indicated by the electronic gauge reaches the minimum thoroughly following each use to protect it from damage. pressure attainable by the system, as no more water vapor is leaving the product. As the heat input to the product is increased, evaporative cooling keeps the product temperature well Backfilling below the temperature of its surrounding atmosphere. When primary drying is complete, the product temperature rises to equal the temperature of its For many freeze dried products, the most ideal system environment. In manifold systems and tray dryers with of closure is while under vacuum. This provides an external collectors, the path to the collector can be shut environment in which moisture and oxygen, both off with a valve and the pressure above the product detrimental to the freeze dried material, are prevented measured with a vacuum gauge. If drying is still from coming in contact with the product. In some cases, occurring, the pressure in the system increases. vacuum in a container may be less than ideal, especially when a syringe is used to recover the product, or when opening the vessel results in a rush of potentially contaminating air. In these cases, backfilling the product Contamination in a container with an inert gas such as argon or nitrogen is often beneficial. The inert gas must be ultrapure, Freeze Dry System containing no oxygen or moisture. Backfilling of the product container is generally useful Two types of contamination can occur in a freeze dry in a batch tray dryer type system. The backfilling should system. One results from freeze drying microorganisms also be carried out through a bacteriological filter. It is and the other results from freeze drying corrosive important that the gas flow during backfilling be slow materials. enough to allow cooling of the gas to prevent raising the The potential for contamination of a freeze drying collector temperature. Backfilling can be carried out to system by microorganisms is real in any system where any desired pressure in those tray dryers that have microorganisms are freeze dried without a protective internal stoppering capability, and the vials then barrier such as a bacteriological filter. Contamination is stoppered at the desired pressure. most evident in batch tray dryer systems where large 8 Stability of Freeze Dried Products

Several factors can affect the stability of freeze dried material. Two of the most important are moisture and oxygen. All freeze dried products have a small amount of moisture remaining in them termed residual moisture. The amount of moisture remaining in the material depends on the nature of the product and the length of secondary drying. Residual moisture can be measured by several means: chemically, chromatographically, manometrically or gravimetrically. It is expressed as a weight percentage of the total weight of the dried product. Residual moisture values range from <1% to 3% for most products. By their nature, freeze dried materials are hygroscopic and exposure to moisture during storage can destabilize the product. Packaging used for freeze dried materials must be impermeable to atmospheric moisture. Storing products in low humidity environments can reduce the risk of degradation by exposure to moisture. Oxygen is also detrimental to the stability of most freeze dried material so the packaging used must also be impermeable to air. The detrimental effects of oxygen and moisture are Contact Labconco for a complimentary catalog of Freeze temperature dependent. The higher the storage Dry Systems designed for laboratory use. temperature, the faster a product degrades. Most freeze dried products can be maintained at refrigerator temperatures, i.e. 4-8° C. Placing freeze dried products at lower temperatures extends their shelf life. The shelf life The Labconco catalog provides additional information of a freeze dried product can be predicted by measuring about freeze drying equipment. It is available upon the rate of degradation of the product at an elevated request from your laboratory supply dealer or Labconco temperature. This is called accelerated storage. By Corporation at (800) 821-5525, (816) 333-8811 or choosing the proper time and temperature relationships www.labconco.com. at elevated temperatures, the rate of product degradation can be predicted at lower storage temperatures.

9 Isothermal Desorption: The process of desorbing water from a Glossary freeze dried product by applying heat under vacuum. Lyophilization: The freeze drying process. Accelerated Storage: Exposure of freeze dried products to elevated temperatures to accelerate the degradation process that Manifold Freeze Drying: A freeze drying process where each occurs during storage. vessel is individually attached to a manifold port resulting in a direct path to the collector for each vessel. Batch Freeze Drying: Freeze drying multiple samples of the same product in similar sized vessels at the same time in a shelf Prefreezing: The process of cooling a product to below its tray dryer. eutectic temperature prior to freeze drying. Bulk Freeze Drying: Freeze drying a large sample of a single Pressure Gauge (Vacuum Gauge): An instrument used to product in one vessel such as the bulk drying pans designed for measure very low pressures in a freeze drying system. shelf tray dryers. Thermocouple Gauge: A pressure gauge that measures only Collapse: A phenomenon causing collapse of the structural the condensable gases in the system. This gauge can be used integrity of a freeze dried product due to too high a temperature as an indicator of drying end points. at the drying front. McLeod Gauge: A mercury gauge used to measure total Collapse Temperature: The temperature above which pressure in the system (i.e. condensable and non- collapse occurs. condensable gases.) Collector: A cold trap designed to condense the water vapor Primary Drying: The process of removing all unbound flowing from a product undergoing freeze drying. water that has formed ice crystals in a product undergoing freeze drying. Internal Collector: A collector located in the same area as the product. All water vapor has a free path to the collector. Residual Moisture: The small amount of bound water that remains in a freeze dried product after primary drying. Residual External Collector: A collector located outside the product moisture is expressed as the weight percentage of water area connected by a small port through which all water vapor remaining compared to the total weight of the dried product. must pass. Allows isolation of the product from the collector The amount of residual moisture in a freeze dried product can for drying end point determinations and easier defrosting. be reduced during secondary drying. Ethylene Oxide: A colorless, odorless gas used for gas Secondary Drying: The process of reducing the amount of sterilization of tray dryer systems. bound water in a freeze dried product after primary drying is Eutectics: Areas of solute concentration that freeze at a lower complete. During secondary drying, heat is applied to the temperature than the surrounding water. Eutectics can occur at product under very low pressures. several different temperatures depending on the complexity of Shell Freezing: Freezing a product in a thin layer that coats the the product. inside of the product container. Shell freezing is accomplished Eutectic Temperature: The temperature at which all areas of by swirling or rotating the product container in a low concentrated solute are frozen. temperature bath. Evaporative Cooling: Cooling of a liquid at reduced pressures Sublimation: The conversion of water from the solid state (ice) caused by loss of the latent heat of evaporation. directly to the gaseous state (water vapor) without going through the liquid state. Freeze Drying: The process of drying a frozen product by creating conditions for sublimation of ice directly to Vapor Pressure: The pressure of the vapor in equilibrium with water vapor. the sample. Glass Transition Temperature: The temperature at which certain products go from a liquid to a vitreous solid without ice crystal formation.

10 21. Rowe, T.W.G. 1970. Freeze-drying of biological materials: Bibliography some physical and engineering aspects. Current Trends in Cryobiology: 61-138. 1. Barbaree, J.M. and A. Sanchez. 1982. Cross-contamination 22. Seligman, E.B. and J.F. Farber. 1971. Freeze-drying and during lyophilization. Cryobiology 19:443-447. residual moisture. Cryobiology 8:138-144. 2. Barbaree, J.M., A. Sanchez and G.N. Sanden. 1985. Problems in freeze-drying: I. Stability in glass-sealed rubber stoppered vials. Developments in Industrial Microbiology 26:397-405. 3. Barbaree, J.M., A. Sanchez and G.N. Sanden. 1985. Problems in freeze-drying: II. Cross-contamination during lyophilization. Developments in Industrial Microbiology 26:407-409. 4. Flink, J.M. and Knudsen, H. 1983. An Introduction to Freeze Drying. Strandberg Bogtryk/Offset, Denmark. 5. Flosdorf, E.W. 1949. Freeze-Drying. Reinhold Publishing Corporation, New York. 6. Greiff, D. 1971. Protein structure and freeze-drying: the effects of residual moisture and gases. Cryobiology 8:145- 152. 7. Greiff, D. and W.A. Rightsel. 1965. An accelerated storage test for predicting the stability of suspensions of measles virus dried by sublimation in vacuum. Journal of Immunology 94:395-400. 8. Greaves, R.I.N., J. Nagington, and T.D. Kellaway. 1963. Preservation of living cells by freezing and by drying. Federation Proceedings 22:90-93. 9. Harris, R.J.C., Ed. 1954. Biological Applications of Freezing and Drying. Academic Press, New York. 10. Heckly, R.J. 1961. Preservation of bacteria by lyophilization. Advances in Applied Microbiology 3:1-76. 11. Heckly, R.J. 1985. Principles of preserving bacteria by freeze-drying. Developments in Industrial Microbiology 26:379-395. 12. Jennings, Thomas. 2002. Lyophilization: Introduction and Basic Principles. CRC Press, Florida. 13. King, C.J. 1971. Freeze-Drying of Foods. CRC Press, Cleveland. 14. May, M.C. E. Grim, R.M. Wheeler and J. West. 1982. Determination of residual moisture in freeze-dried viral vaccines: Karl Fischer, gravimetric, and thermogravimetric methodologies. Journal of Biological Standardization 10:249-259. 15. Mellor, J.D. 1978. Fundamentals of Freeze-Drying. Academic Press, London. 16. Nail, S.L. 1980. The effect of chamber pressure on heat transfer in the freeze-drying of parental solutions. Journal of the Parental Drug Association 34:358-368. 17. Nicholson, A.E. 1977. Predicting stability of lyophilized products. Developments in Biological Standardization 36:69-75. 18. Parkes, A.S., and A.U. Smith, Eds. 1960. Recent Research in Freezing and Freeze-Drying. Charles C. Thomas, Springfield. 19. Rey, L.R., Ed. 1960. Traite de Lyophilization. Hermann, Paris. 20. Rey, L.R., Ed. 1964. Aspects Theorique et Industriels de la Lyophilisation. Hermann, Paris.

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